DNase Test Principle and Procedure

In this DNase test principle and procedure post we have briefly explained about DNase test principle, objectives, requirements, procedure, uses and limitations of DNase test.

DNase Test Principle and Procedure

DNase test is used to assess an organism’s capacity to hydrolyze DNA. DNase test agar is a differential media that evaluates an organism’s ability to produce the exo-enzyme deoxyribonuclease.

DNase test agar, a differential medium, is used to assess an organism’s ability to make deoxyribonuclease, or DNase. The DNase test is used to detect deoxyribonuclease activity in bacteria and fungi, and is especially useful for identifying pathogenic Staphylococci.

It can also be used to differentiate Serratia spp. from Enterobacter spp., Staphylococcus aureus from other species, and Moraxella catarrhalis from Neisseria sp.


DNase test Agar is used to detect deoxyribonuclease activity in bacteria and fungi, and to identify pathogenic Staphylococci in particular.

It is employed in the distinction and identification of non-pigmented Serratia species isolated from clinical sources that may be mistakenly recognised as Enterobacter and Klebsiella species with the addition of toluidine blue.

Weckman and Catlin were the first to investigate the relationship between DNase activity and coagulase activity. Jeffries et al used an agar plate approach using a semi-synthetic medium to demonstrate DNase activity.

When the plates were soaked with 1 N hydrochloric acid, positive DNase activity was seen as clear zones (around colonies). By introducing DNA into the medium along with calcium chloride to activate the enzyme, DiSalvo was able to validate the link between coagulase activity and DNase activity.

To activate DNase enzyme, Di Salvo combined DNA with calcium chloride. Toluidine blue was added to the DNase test medium by Schreier. Serratia marcescens was identified faster using this modified medium, which also allowed Serratia to be distinguished from other Enterobacteriaceae members.

No Indicator Dye

The hydrolysis of DNA is noticed in DNase agar without an indicator by a clearing of the agar after the addition of HCL (oligonucleotides dissolve in acid but DNA salts are insoluble).

The acid causes unhydrolyzed DNA to precipitate, making the medium opaque. As a result, colonies that produce DNase hydrolyze DNA and create a clean zone around the growth.

Toluidine Blue O

TBO (Toluidine Blue O) is a metachromatic dye that changes colour when it is complexed with various substances. TBO produces a royal blue tint when it combines with polymerized DNA (uninoculated medium).

TBO forms a compound with oligonucleotides or mononucleotides when DNA is hydrolyzed, resulting in a change in dye structure and absorption spectrum, resulting in a brilliant pink colour. On a blue background, DNase-positive colonies emerge with rose-pink halos.

When evaluating Campylobacter species, Lior and Patel suggested using DNase Agar with TBO. It’s also worth noting that TBO may suppress several gram-positive bacteria, including some forms of Staphylococcus aureus.

Methyl Green Dye

Methyl green dye is used as an indication on the DNase test agar plate in this case. Methyl green intercalates (binds) between the bases of double-stranded DNA with an association constant of 9×10-5 M-1 for normal DNA and (1×10-5 M-1 for denatured DNA.

A maximum of 5 base pairs of native double-stranded DNA can bind a maximum number of dye molecules. Methyl Green binds to DNA and forms a complex; the methyl green-DNA complex is green in colour when formed at pH 7.3 or neutral. Methyl green, on the other hand, remains colourless at this pH.

When deoxyribonuclease (deoxyribonuclease) performs enzymatic hydrolysis of the phosphodiester link and breaks the DNA molecules, free methyl green molecules are produced. The fact that free methyl green decolorized spontaneously at pH 7.3 or neutral is most likely due to dye tautomerization.

As a result of the ability of DNase test activity produced by the test Organism, the zone of clearance on solid media and reductions in colour intensity in liquid media are seen.



1. Sterile sticks

2. Inoculating loops

3. Pasteur pipettes

4. Boiling heat block

5. Incubators at 35 and 30°C


1. 1 N HCl

Media Composition

1. Pancreatic digest of casein (10 g), yeast extract (10 g), deoxyribonucleic acid (2 g), NaCl (5 g), agar (15 g), methyl green or Toluidine Blue O (0.5 g), pH 7.5.

Media preparation

42 grams of dehydrated powder or lab-prepared media are combined with 1000 millilitres of pure distilled or deionized water in a beaker.

To completely dissolve the medium, the suspension is heated with agitation to bring it to a boil. The dissolved medium is then autoclaved for 15 minutes at 15 lbs pressure (121°C).

After the autoclaving procedure is completed, the beaker is removed and chilled to around 40-45°C.

Under sterile circumstances, the medium is poured into sterile Petri plates. If you want to use indicators, add 0.1 gram of toluidine O or methyl green to the medium before sterilising it.


1. Streak a loop full of culture from a previously treated test organism plate onto a sterile DNase test medium plate with the respective indicator dye.

2. Then, at 37° C, incubate this plate overnight or for 24 hours under aerobic conditions.

3. If you’re using indicator dye, look for a colour shift or a zone of clearance after incubation.

4. In the case of DNase test without indicator. Allow the reagent to absorb into the agar plate by flooding it with 1N HCl and removing the tip of the access acid from the plate.

5. Allow 5 minutes for the acid to react with the substrate. Keep an eye on the clearance zone around the colonies.


Methyl Green

DNase Test

DNase positive (top).

DNase Positive (+): Clear zones around bacterial streak and colonies against a green background in DNase test. Clear zones are best observed against a white background. 

DNase Negative (−): No color change, medium remains green

Toluidine Blue O

DNase Test

DNase positive (Left).

DNase Positive (+): Bright rose-pink zone around bacterial streak or colonies against a royal blue background. 

DNase Negative (−): No color change, medium remains blue

Without indicator

DNase Test

DNase test of Staphylococcus spp. The strain in the upper streak is negative (no clearing around the streak), whereas the strain in the lower streak is positive

DNase positive (+): DNase positive organisms will be surrounded by clear zones of depolymerized DNA while the medium farther away from the inoculation band will be opaque and whitish due to polymerized DNA.

DNase negative (-):  Colonies of DNase test negative organisms will not show any clearing around the colonies.

Further Readings